The flaviviruses contain many pathogens of public health importance, including dengue virus (DENV) and West Nile virus (WNV) for which we have no licensed vaccines. Live attenuated vaccines, which induce durable protective immunity, are one of the important strategies to control flavivirus diseases and have been very successful at controlling yellow fever (YF) and Japanese encephalitis (JE). The nonstructural (NS) proteins are associated with evasion of host innate immunity. Among them, NS4B protein has extensive homology between flaviviruses and the N-terminal region has been shown to encode a major interferon (IFN) antagonism function. Thus, we focus on NS4B to identify stable mutations that can be used to rationally attenuate candidate live flavivirus vaccines. We are using WNV as a model and have identified two highly attenuated WNV NS4B mutants. The C102S mutant has a substitution in the flavivirus conserved central hydrophobic domain of NS4B protein, which is known to contribute to the virulence phenotype of flaviviruses, and contains mutations in the attenuated derivatives of YFV, JEV and DENV. Although the NS4B-C102S mutant was highly attenuated for mouse neuroinvasiveness and neurovirulence, and conferred protective immunity in mice, it was prone to reversion. It is not an ideal candidate vaccine by itself. Another attenuated WNV mutant has a substitution in the highly conserved P38 residue in the N-terminal region of the NS4B protein. It is highly attenuated for mouse neuroinvasiveness, highly immunogenic, reversion of the mutant has not been identified. Our recent published work showed that the NS4B-P38G mutant induced higher innate cytokine and memory T cell responses in mice than the wild-type (WT) NY99 strain, and immunized mice were all protected from WT WNV challenge. The underlying immune mechanisms are not clearly understood. We hypothesize that the P38G substitution in the IFN antagonism region or in combination with mutation(s) in the conserved central hydrophobic region of WNV NS4B protein will confer an attenuated phenotype and the ability to induce higher protective immunity than WT NY99, and this may serve as an excellent model to develop potential live flavivirus vaccine candidate(s). We will first elucidate the immune mechanisms by which WNV NS4B-P38G mutant triggers higher protective immunity than WT NY99. We will next determine whether incorporating the NS4B-C102S mutation will further enhance and stabilize the attenuated phenotype of WNV NS4B-P38G mutant. Lastly, we will characterize immune responses to WNV NS4B-P38G/C102S double mutant infection in mouse and human cells and evaluate the protective effects of WNV NS4B mutant strains after immunization of mice followed by lethal WT WNV challenge. Characterization of stable mutations in the highly conserved coding regions of WNV NS4B protein and investigation the mechanism by which WNV NS4B mutant induces higher protective immunity can be utilized as a paradigm to aid in the rational development of other efficacious live attenuated flavivirus vaccines.